Agonist stimulation, talin-1, and kindlin-3 are crucial for α(IIb)ß(3) activation in a human megakaryoblastic cell line, CMK.

Platelet integrin α(IIb)ß(3) activation is regulated by inside-out signaling via agonist stimulation. However, when α(IIb)ß(3) was exogenously expressed in cell lines such as Chinese hamster ovarian cells, physiological agonists hardly induced α(IIb)ß(3) activation. To overcome this disadvantage, we characterized the functional regulation of endogenously expressed α(IIb)ß(3) in a megakaryoblastic cell line, CMK, employing an initial velocity assay for PAC-1 binding. We firstly demonstrated that protease-activated receptor 1-activating peptide induced robust, but transient α(IIb)ß(3) activation in CMK cells with high glycoprotein-Ib expression. Stable talin-1 or kindlin-3 knockdown cells confirmed that the protease-activated receptor 1-activating peptide-induced α(IIb)ß(3) activation was dependent on talin-1 and kindlin-3 expression. In sharp contrast to exogenously expressed α(IIb)ß(3) in Chinese hamster ovarian cells, transient overexpression of full-length talin (FL-talin) or talin-head domain (THD) alone did not activate α(IIb)ß(3) in CMK cells, but required agonist stimulation. Similarly, kindlin-3 overexpression alone did not induce α(IIb)ß(3) activation, but it significantly augmented agonist-induced α(IIb)ß(3) activation. Several mutants of FL-talin and THD suggested that the head-rod interaction was critical for autoinhibition of talin-1, and the interaction between the THD and the membrane-proximal region of the ß(3) cytoplasmic tail was essential for talin-mediated α(IIb)ß(3) activation. In addition, THD and kindlin-3 cooperatively augmented protease-activated receptor 1-induced α(IIb)ß(3) activation. We proposed that the CMK cell line is an attractive platform for investigating agonist-, talin-1-, and kindlin-3- dependent α(IIb)ß(3) activation.

Recognition of highly restricted regions in the ß-propeller domain of αIIb by platelet-associated anti-αIIbß3 autoantibodies in primary immune thrombocytopenia.

Platelet-associated (PA) IgG autoantibodies play an essential role in primary immune thrombocytopenia (ITP). However, little is known about the epitopes of these Abs. This study aimed to identify critical binding regions for PA anti-αIIbß3 Abs. Because PA anti-αIIbß3 Abs bound poorly to mouse αIIbß3, we created human-mouse chimera constructs. We first examined 76 platelet eluates obtained from patients with primary ITP. Of these, 26 harbored PA anti-αIIbß3 Abs (34%). Further analysis of 15 patients who provided sufficient materials showed that the epitopes of these Abs were mainly localized in the N-terminal half of the ß-propeller domain in αIIb (L1-W235). We could identify 3 main recognition sites in the region; 2 eluates recognized a conformation formed by the W1:1-2 and W2:3-4 loops, 5 recognized W1:2-3, and 4 recognized W3:4-1. The remaining 4 eluates could not be defined by the binding sites. Within these regions, we identified residues critical for binding, including S29 and R32 in W1:1-2; G44 and P45 in W1:2-3; and P135, E136, and R139 in W2:3-4. Of 11 eluates whose recognition sites were identified, 5 clearly showed restricted κ/λ-chain usage. These results suggested that PA anti-αIIbß3 Abs in primary ITP tended to recognize highly restricted regions of αIIb with clonality.

A potential role for α-actinin in inside-out αIIbß3 signaling.

Many different biochemical signaling pathways regulate integrin activation through the integrin cytoplasmic tail. Here, we describe a new role for α-actinin in inside-out integrin activation. In resting human platelets, α-actinin was associated with αIIbß3, whereas inside-out signaling (αIIbß3 activation signals) from protease-activated receptors (PARs) dephosphorylated and dissociated α-actinin from αIIbß3. We evaluated the time-dependent changes of the αIIbß3 activation state by measuring PAC-1 binding velocity. The initial velocity analysis clearly showed that PAR1-activating peptide stimulation induced only transient αIIbß3 activation, whereas PAR4-activating peptide induced long-lasting αIIbß3 activation. When αIIbß3 activation signaling dwindled, α-actinin became rephosphorylated and reassociated with αIIbß3. Compared with control platelets, the dissociation of α-actinin from αIIbß3 was only transient in PAR4-stimulated P2Y(12)-deficient platelets in which the sustained αIIbß3 activation was markedly impaired. Overexpression of wild-type α-actinin, but not the mutant Y12F α-actinin, increased its binding to αIIbß3 and inhibited PAR1-induced initial αIIbß3 activation in the human megakaryoblastic cell line, CMK. In contrast, knockdown of α-actinin augmented PAR-induced αIIbß3 activation in CMK. These observations suggest that α-actinin might play a potential role in setting integrins to a default low-affinity ligand-binding state in resting platelets and regulating αIIbß3 activation by inside-out signaling.

Molecular analysis of a patient with type I Glanzmann thrombasthenia and clinical impact of the presence of anti-αIIbß3 alloantibodies.

The occurrence of transfusion-related alloimmunization against αIIbß3 is a major concern in patients with Glanzmann thrombasthenia (GT). However, few data are available about molecular defects of GT patients with anti-αIIbß3 alloantibodies as well as clinical impact of these antibodies on platelet transfusion. Here, we report a case of type I GT with anti-HLA and anti-αIIbß3 alloantibodies, who underwent laparoscopic total gastrectomy due to gastric cancer. We found a novel ß3 nonsense mutation, 892C > T (Arg272X), and the patient was homozygous for the mutation. Laparoscopic gastrectomy was successfully performed with continuous infusion of HLA-matched platelet concentrates and bolus injection of recombinant factor VIIa at 2 h intervals. Total bleeding was 370 mL and no red-cell transfusion was necessary. Flow cytometric analysis employing anti-αIIbß3 monoclonal antibody revealed that the transfused platelet count was maintained around 20-30 × 109/L during the operation and 10 × 109/L on the following day. Flow cytometric analysis also showed that transfused platelets retained normal reactivity to ADP stimulation. These results indicate that flow cytometry is useful to assess survival and function of transfused platelets in GT patients with anti-αIIbß3 antibodies.

Antifungal activity of micafungin in serum.

We have evaluated the antifungal activity of micafungin in serum by using the disk diffusion method with serum-free and serum-added micafungin standard curves. Serum samples from micafungin-treated patients have been shown to exhibit adequate antifungal activity, which was in proportion to both the applied dose and the actual concentration of micafungin measured by high-performance liquid chromatography. The antifungal activity of micafungin in serum was also confirmed with the broth microdilution method.

Donor cell-derived chronic myeloproliferative disease with t(7;11)(p15;p15) after cord blood transplantation in a patient with Philadelphia chromosome-positive acute lymphoblastic leukemia.

We report a case of donor cell-derived chronic myeloproliferative disease with t(7;11)(p15;p15) occurring after cord blood transplantation (CBT). A 41-year-old man developed precursor B-cell acute lymphoblastic leukemia with a karyotype of 46, XY, t(9;22)(q34;q11) and inv(9)(p11;q13), for which he received CBT from a sex-mismatched donor at the first complete remission of the leukemia. Five months after CBT, gradual neutrophilia of unknown origin developed following the myeloid reconstitution after CBT. Karyotyping of bone marrow cells at 9 months after CBT showed 46, XX, t(7;11)(p15;p15) in 17/20 dividing cells, but neither Philadelphia chromosome (Ph) nor inv(9)(p11;q13) was present. This is the first report of chronic myeloproliferative disease with t(7;11)(p15;p15) that developed in donor cells after CBT. The donor was well-developed and healthy, at least at the time of follow-up, half a year after the birth.

Donor Cell-Derived Chronic Myeloproliferative Disease with t(7;11)(p15;p15) after Cord Blood Transplantation in a Patient with Philadelphia Chromosome-Positive Acute Lymphoblastic Leukemia.

We report a case of donor cell-derived chronic myeloproliferative disease with t(7;11)(p15;p15) occurring after cord blood transplantation (CBT). A 41-year-old man developed precursor B-cell acute lymphoblastic leukemia with a karyotype of 46, XY, t(9;22)(q34;q11) and inv(9)(p11;q13), for which he received CBT from a sex-mismatched donor at the first complete remission of the leukemia. Five months after CBT, gradual neutrophilia of unknown origin developed following the myeloid reconstitution after CBT. Karyotyping of bone marrow cells at 9 months after CBT showed 46,XX, t(7;11)(p15;p15) in 17/20 dividing cells, but neither Philadelphia chromosome (Ph) nor inv(9)(p11;q13) was present. This is the first report of chronic myeloproliferative disease with t(7;11)(p15;p15) that developed in donor cells after CBT. The donor was well-developed and healthy, at least at the time of follow-up, half a year after the birth.

A novel chromosomal abnormality, t(6;10)(q27;q22), found in a polycythemic potential donor for allogeneic hematopoietic stem cell transplantation.

A 59-year-old man was a potential donor for allogeneic hematopoietic stem cell transplantation and was found to be healthy but slightly polycythemic. The bone marrow was morphologically normal, but karyotyping of bone marrow cells showed t(6;10)(q27;q22) in 7 of 20 metaphases analyzed by G-banding and only the t(6;10) abnormality in 3 of 5 metaphases analyzed by the spectral karyotyping method. G-banding analysis of peripheral blood mononuclear cells cultured with phytohemagglutinin for 72 hours showed a normal karyotype in all 20 metaphases analyzed.These findings suggest clonal expansion with t(6;10)(q27;q22) in the bone marrow of this individual. He was determined to be ineligible for donation. A coordinated nationwide work-up for older donors is necessary to ensure high-quality standards.